Humoral immunity relies on the specific recognition and elimination of foreign antigen (Ag), brought about by immunoglobulin (Ig) Ag receptors present on B cells. The humoral immune system needs a vast and diverse array of antibody (Ab) molecules to specifically recognize the extensive and varied world of potential Ags. It achieves this in part by encoding a large array of germline variable (V), diversity (D) and joining (J) gene segments. The germline, junctional and combinatorial diversity, together with Ag dependent changes such as somatic hypermutation and receptor editing, collectively produce a potentially vast diversity of V gene sequences.
The specialized microenvironments present in secondary lymphoid organs like the spleen and lymph nodes, where T-cell dependent Ab responses occur, are referred to as germinal centers. In germinal centers, Ag specific B cells undergo clonal expansion, somatic hypermutation, isotype class switching, affinity maturation, Ag-driven selection and differentiation into memory B cells or plasma cells.
PCR technology has been used for studying various aspects of B cell biology such as B cell repertoire analysis, expression of Ab using phage display systems and detection of B cell clonality in diagnostic haematopathology. Using PCR technology, numerous investigators have tried to devise a universal primer or set of primers for amplifying all possible mouse V gene rearrangements (Chiang et al., 1989; LeBoeuf et al., 1989; Orlandi et al., 1989; Larrick et al., 1990; Coloma et al., 1991; Kettleborough et al., 1993; Nicholls et al., 1993; Zhou et al., 1994; Dattamajumdar et al., 1996; Lan et al., 1996; Krebber et al., 1997). These primers, degenerate or otherwise, were however not successful in amplifying all the V gene segments (Owens and Young, 1994; Ruberti et al., 1994; Wang et al., 2006). Several workers have tried to overcome the shortcomings of the earlier efforts by designing a few, highly degenerate (1- to 128-fold or more degeneracy) primers (Jones and Bendig, 1991; Wang et al., 2000; Essono et al., 2003). In general, highly degenerate primers have higher propensity to lead to cross-family amplification. An alternative approach involves designing a panel of primers with minimum degeneracy (Chardes et al., 1999; Seijen et al., 2001). These studies (Chiang et al., 1989; LeBoeuf et al., 1989; Orlandi et al., 1989; Larrick et al., 1990; Coloma et al., 1991; Jones and Bendig, 1991; Kettleborough et al., 1993; Nicholls et al., 1993; Owens and Young, 1994; Ruberti et al., 1994; Zhou et al., 1994; Dattamajumdar et al., 1996; Lan et al., 1996; Krebber et al., 1997; Chardes et al., 1999; Wang et al., 2000; Seijen et al., 2001; Essono et al., 2003; Wang et al., 2006) designed primers for a standard PCR. Further, they do not report the PCR detection limit achieved using their primer set which is unlikely to be sensitive enough to be able to amplify the rearranged VH and VL genes from a single or a small number of B cells. To enhance the sensitivity and specificity of the PCR for analysis of the rearranged VH and VL genes from single B cells, researchers working on the human and rabbit systems have resorted to a nested PCR strategy (Brezinschek et al., 1995; Foster et al., 1997; Sehgal et al., 1998; Farner et al., 1999; Dohmen et al., 2005).
The major challenge in using PCR technology for studying mouse Ab genes, whether expressed in homogeneous hybridoma cell lines or a heterogeneous splenic B cell population, is to ensure broad and highly specific amplification. This need arises due to the fact that the mouse V genes show intra- and inter-family sequence variability, and somatically rearrange to generate nearly limitless Ab diversity. Additionally, somatic hypermutation events that coincide with the primer binding sites can potentially adversely affect the efficiency of amplification.
In order to study V gene usage, somatic hypermutation patterns, clonal expansion and selection in Ag-specific splenic B cells in experimental system, one would require a PCR primer set and an amplification strategy that would potentially amplify all possible rearranged V genes from a single or a small number of B cells in a highly sensitive and specific manner. A nested primer set that can potentially amplify the rearranged VH and VL genes belonging to any V gene family from single or small number of mouse B cells in a representative and highly specific manner has not been reported.
The present invention discloses a novel nested PCR primer set that amplifies all possible mouse rearranged/expressed VH, Vκ and Vλ genes from C57BL/6 and BALB/c mice in an isotype-independent manner with minimum cross reactivity, low degeneracy, high specificity and sensitivity, using genomic DNA or total RNA from small number of B cells.